Snake Venom Toxin Evolution: Venom Gland Diversification

[00:00:18]so hi everyone and thanks for being interested in watching this amazing lecture and that's about to come and we have today here professor stephen maxey from the school of biological science at the university of northern colorado his talk is going to talk about the evolution and diversification of his snake venom toxins and venom glands and it's a great pleasure to to have you here professor stephen and on behalf of the the graduate students and research office of unifies we would like to thank you but we also would like to thank you professor miriam professor miro is a a tremendous partner of the the graduate studies and research office she's been dedicating her time to organize a lot of international webinars and for sure today it's going to be another one with a very successful interaction between our inviting in our our public and professor doctor miriam hayashi she was born and educated in brazil and he's currently associated professor of pharmacology at univespy and head of the molecular pharmacology lab at the department of pharmacology at the scala paulista de medicina professor hayashi is also vice chief of the discipline in cellular pharmacology and member of the scientific board of the intellectual property office of university she completely uh her pharmacy and biochemistry degree at the state university of londorina in parana and she received her phd at unifesti and post doctoral fellowship in molecular biology at bhutan institute which is a brazilian institution mostly renowned by the scientific research with venomous animals and productions of vaccines and the dr hayash has published more than 90 scientific articles on mental disorders and natural compounds for drugs discovered feud besides several chapters of books and patent applications filed and she's also is a uh was she was also recently awarded with the the prize from theory group innovation award and thanks to development of a method of identifying biomarkers for several for severe mental illness by nuclear magnet resonance and chemometrics and so she is a very important and today we have a very important topic to discuss here because it's a topic in between our the most prestigious area of unifest which is the health area but also we have a lot of biodiversity researchers and i'm pretty sure our students our post graduation students and grad students they are here for listen to this amazing thank you so much to both of you professor miriam and professor stevens welcome thank you camille for this great um introduction my wife your first doctor stephen so first of all thank you very much for accepting these invitations uh dr stephen mckissey is a professor of biology and school of biological science at the university of northern colorado he such broadly encompasses the biology of venom snake and the biochemistry of snake venos and he has published more than 200 scientific papers with captors and natural history notes including several books one of them is is the reason that we know each other let's name it the handbook of book uh and toxin of reptiles crc press so if you are interesting please buy this book it's called which is a new one new magician this year and this his research has included many graduate and undergraduate students as well as a collaboration with colleagues from singapore spain mexico costa rica argentina brazil france india and others including other universities in the usa and university of texas several ongoing projects are centered on understanding the evolution of feminine system is next and the biological significance of venom composition of variation particularly in areas of introgression and hybridization with a special interesting interface between snake ecology evolution and venom biochemistry and pharmacology so uh dr stephen sorry for my author english and uh i hope i could just highlight the main achievements your cv is really great and long so he would stay like half an hour reading it in my in my bad english so i will give the screen for you and thank you again for your invitation well it's very much my pleasure to be here i wish i was there in person rather than electronically but this is our life these days so i'd like to thank in particular professor hayashi for the invitation to talk to you today about some of the work that we do in my lab and some of the things i'm going to talk about today started a long time ago and have extended into recent times as well so hopefully you'll find some interesting things as we go through talking about evolution and diversification of venoms and venom glands and excuse me if i constantly am clear in my throat sorry if i interrupt you i forgot just giving advertise for the attendees so uh you can make questions in the chat so you write the question and you can read the questions for dr stephen that is going to answer the at the end of this presentation okay thank you i'm sorry go ahead no no no problem yeah please do ask ask anything and i'll give my best attempt to try to answer it um before we get started though i thought i'd show you a little bit about what things are like right here because colorado is a bit different from sao paulo at the moment and let's see if we can so this is what we're looking at today in colorado and it's supposed to be spring but yesterday a snowstorm came in and coated everything with snow so even though we're going into spring we still have vestiges of winter and it was quite cold this morning so anyhow i get this question a lot why study venoms and to me it's kind of a strange question on non-sequitur of course you should study venoms they're very interesting but if we think about it venoms are these very potent biological products that have toxic effects and so that of course just in itself is rather interesting if you're more evolution and ecologically minded these are characteristics that have direct fitness components as well as implications and so if you think about that snakes are producing these toxins they're mobilizing their prey with it and so they're obtaining food by using these remote delivered chemical means of predation we can also look at this from a chemical predatory sort of strategy and ask questions about why venoms vary from this snake to that snake why are coral snakes and rattlesnakes so very different in their venom composition and along that line that provides an open area for all sorts of potentials for drug discovery and interesting new pharmaceutical compounds that may in fact have therapeutic applications so if you're more interested in esoteric biochemistry protein evolution and aspects of how toxins in fact have evolved over time developed new activities neo-functionalization of these molecules is also an area and one thing i'll talk a little bit about today is structure function type of studies so how we can have these molecules that on the surface look very similar but small changes lead to very large changes in their biological activities so what i'd like to do is start out giving you a rather extended overview of venomous snakes talk a little bit about the diversity of species that we see worldwide a little bit about the venom apparatus that is used to produce and deliver these compounds and then talk a bit about what we can call venom compositional strategies okay to do this i'll use two vignettes from work that we have been conducting over some time really and some of the earliest of this started 20 years ago and has extended until current times so we'll really look at a kind of a combination of features that provide an integrative biology approach to things and then talk about three-finger toxins a particular family of compounds with some very interesting properties okay finally then i'll try to wrap this up summarize and leave you with some take-home conclusions okay so first let's take a look at venomous snakes and their venom apparatus and so just to give you a feel for the diversity of the actors in this whole scene of what we call toxinology the study of venoms there are a wide variety of different types so we've got old world vipers that range in size from very large to very small body we have old world pit vipers that are characterized by heat sensitive pit organs a very diverse group of these all through asia we have in the new world in sometimes in in your area your general vicinity in in that part of brazil a wide variety of pit vipers that are highly specialized as arboreal snakes or terrestrial snakes in the new world we have also a wide diversity of rattlesnakes and these reach their apex in mexico but they extend all the way from canada into south central um south america so very broad range and very interesting diversity of species and a lot of work that we've done has included these various species of rattlesnakes again some of them highly specialize all these are mountain forms and then there are lowland tropical rattlesnakes that have much larger body form this contrasts rather distinctly with the lapid snakes things like cobras the coral snakes and their allies and very very different venoms we'll talk about those briefly but we're going to spend a bit of time talking about rear fang snakes in several different families including the columbra day and they also are very very diverse in their form and their distribution globally their habits and their basic biology and so another family is the just dip saturday and i'm not going to belabor the taxonomy because higher snake taxonomy has been a point of contention for many many years for many people suffice it to say there's quite a diversity of forms and so what we see in this is a cladogram that was published several years ago by other people and in fact what we have is a wide diversity of different types of species globally if we ask the question where do we find venoms well the vipers as we've just seen are reasonably group diverse group of animals that range all the way from the tropics to the subtropics in temperate regions the elapids more of a tropical subtropical group and there's a fair diversity of species here at three four three or four hundred species total when we look at the so-called pollutants or the rear fang snakes there is a tremendous diversity of species and the majority of advanced snakes fall into these two families groups three family groups depending on how you wanna person so we have a lot of different species potentially to work with and in particular this group is much less worked on than the vipers and the alapins so if you're interested in any aspect of biology there is a particular sub discipline of biology that is involved in the study of venoms and so venoms have become a very popular area for research regardless of whether you're more morphology based comparative anatomy type of things where you're looking at the structure of the glands themselves again if you're more evolution ecology based or if you're really focused in on protein chemistry the venoms are primarily proteins so lots to do in that area proteomics transcriptomics and genomics have of course blossomed and and taken the stage front stage in many areas of biology and of course these are very very important disciplines for understanding venom's venom pollution and venom structure function relationships so regardless of where your interest in biology are there's probably some niche there that includes things like the study of venoms okay one of the things that i was first interested in as a graduate student and then still continue to this day is looking at the structures themselves front fang snakes such as rattlesnakes and genus crotalus have a very large reservoir of venom in this venom gland it is delivered with a very high pressure at a very rapid rate and so the whole strike sequence strike and venomation and retract takes somewhere on the order of 250 milliseconds very very fast delivered through these teeth that are hollow and are very much like a hypodermic syringe needle if we contrast that with the rear fang snakes we have a much lower pressure system and this is delivered via a gland that has similarities to the venom gland of front fang snakes but typically lacks any kind of muscular push to deliver the venom at a rapid rate so it's a relatively slow low pressure delivery system that's associated with enlarged teeth on the back of the maxillary bone here and here we can see that in larger detail it's still very distinctive histologically from the salivary glands that are muco secret story and these are cirrus secretory producing the venom proteins that also affects how we get the venom from the snakes front fang snakes relatively easy grab the snake express the venom into a container rear fang snakes even something like this big boom sign here much much slower much more laborious process requires a two drug regimen to get them to secrete significant amounts of material so the venom extraction process is different as well well if we think about what's in the venom as i mentioned it's primarily proteins these are not de novo toxins but instead they're co-opted compounds that are based on regulatory compounds that are found commonly in the body of vertebrate animals and they're over expressed in specialized glands and in conjunction with todd casto's group in university texas arlington we've been very interested in looking at some of the molecular mechanisms of how this is controlled and regulated and up regulated very rapidly so we have a material that is really really potent as far as its biological activity but it's really only contains a handful of representatives of protein families and the thousands of different proteins that are out there there's only about 32 maybe 35 that are commonly seen in venoms and of that a much smaller number that are the most abundant components but snakes have done a lot with those found components and in a particular molecular scaffold may be highly conserved but little tweaks and mutations on the surface features have in fact produced very very different pharmacologies using the same basic molecular scaffold we'll look at an example of that in a little bit here and so by having these non-structural features of the molecule that are basically open to evolution and variation we can produce things that have very different biological activities but structurally are quite similar okay it's been recognized a long time ago now that we have greater variation in the portion of the genes that actually codes for the end molecule rather than in introns and that's kind of opposite from what we see in most standard proteins so another interesting group of compounds now this is the one you'll want to write down because there'll be a quiz at the end you'll have to memorize all these but not really what we see is there are a number of different types of enzymatic toxins and non-enzymatic toxins they're found in venoms but some of them actually predominate and we'll talk a little bit more about some of those in a minute here i like to take venoms and do a fingerprinting with 1dscs page for one for a couple of reasons one it's very easy undergraduates can do this with very little training but two it produces a nice picture of the overall complexity that we may be encountering in a venom that we haven't looked at before so what we have here is a picture of a gel mass ranging from high mass here to low mass here and then on the top here and in each one of these lanes are different species venoms from rattlesnakes of a variety of different types and just a quick glance here we can see that this one contains quite a bit of p1 metalloproteinase whereas these species in here seem to lack that one and so we can get a fair amount of information because we already know quite a bit about where molecules are going to band out on something like a gel well as we've moved into the proteomics era we can use a wide variety of different types of tools mass spectrometry multi-top and esi and a whole host of other different types of technologies have been proliferating very rapidly over the last 10 or 15 years and these allow us to work with extremely minor amounts of venom that has led to this idea of genomics or the study of the venom proteins proteome sorry and we can start with a variety of different types of materials but with very very small amounts and subject this to a number of different experimental pathways and protocols and get different types of detailed information from microgram quantities or less of the venom and this allows us then potentially to catalog the entire proteome there are some drawbacks to some of these techniques and limitations but first approximation this is extremely uh rapid way to go through venoms and get a good feel for what's in the venom at an individual level at population levels and so we can have a deep deep understanding of venom composition and compare that between species quite readily using almost no material at all juan calveri in valencia spain is very important as kind of a central individual that has really developed and pushed the nomics as an area of venom study and so in a paper that he wrote about 14 years ago here he lays out the basic pathway and this is still being used by many people globally to understand venom complexity well as we've gotten into proteomics and and transcriptomics and genomics in more and more detail it turns out that a variety of different approaches will give us the most complete picture of whatever it is we're looking at and that includes venom composition and so using next-gen sequencing and transcriptomics and and genomics approach and a whole host of other methods then we can really get at looking at everything that's possible that can be expressed in the venom gland and so this also favors a high throughput approach where we can deal with lots of samples in a very short period of time it has the added advantage that instead of having fragments of sequence we can in fact get complete sequences of the dna from the genes themselves but also then translate that into the amino acid and again we can address the same kind of questions at individual population levels and broader look at phylogenetic patterns of venom evolution venom composition and so by using these various techniques that again have become commonplace now in most fields of biology we can really probe these systems in much more detail than was ever possible before okay what this allows us to do then is start looking at things and asking the question well do we see some patterns and i'm going to show you some patterns here that have fallen out seemed apparent to me this is not absolute in biology nothing is absolute if there are more than one way to do things efficiently biology finds a way in the viperids though that we've worked with extensively what we see is what we call type one versus type two venoms and there's some very prominent differences between these types of venoms one the type ones tend to be more enzymatically based and result in tissue degradation and all sorts of problems upon human envenomations whereas type 2 venoms typically lack the metalloproteinase complements or it's very very low but it's dominated by presynaptic neurotoxins and if we then ask do we see this kind of pattern in the other groups of venomous snakes to an extent we do see the same kind of dichotomies falling out and again this is a broad brush generalization but the lapids are mostly a type 2 venom that is a highly toxic venom that is very dependent on specific toxins three finger toxins in particular fossil lipase toxins as well and very little in the way of metallic rhodonasis and we've asked the question about the columbian snakes and so what we see is a similar sort of dichotomy in various species so ranging from metalloproteinases that are highly abundant in the venoms and essentially lacking anything that is neurotoxic to having metalloproteinases as only minor or perhaps not even present at all in venom and instead a venom that is dominated by three finger toxins and there's some interesting aspects about these that we'll get to in a moment so just to give you an idea of the picture of this difference in the type 1 type 2. this is just a picture of a composition of various species venoms and again a broad brush comparison here so metalloproteinases serium proteinases phospholiphases dominate this particular venom and we see comparable patterns all the way across the board in these species that are pictured here and these are what we call the type 1 dominated by enzymatic toxins and the scmps in particular contrasting rather strongly with the type 2 that are dominated by things like these beta neurotoxins that are pre-synaptic in their action and in fact some of these such as the rattlesnake that you have down in your area commonly is in fact dominated by these neurotoxic components if we compare those patterns to to a lapids we see a very different sort of picture here and that is the type twos are found across the board but we see some interesting differences in the proportions of three finger toxins and phospholipases in particular but those two protein families dominate the proteomes of all of these different species whether we're talking new world species old world species or these specialized snakes the sea snakes which have a very very simple strip down them taking that approach to the collude we have fewer species to choose from but we see the same kind of dichotomy here a type one enzyme rich sort of venom versus one that is very rich in specific types of toxins specifically three finger toxins okay so these are targeting particular systems in prey animals and in in our cells when we happen to be the recipient of venoms unfortunately one of these is the peripheral nervous system you think about stopping prey hitting the way that the neuromuscular system is coordinated it's probably a good idea and so a lot of different toxins have evolved among these snakes and allow them to knock things out either at a presynaptic site here where we're interfering with acetylcholine release or commonly at a postsynaptic site here where we are binding to the acetylcholine receptor and inhibiting the signal from reaching its target well muscular systems are also targeted by a variety of different types of toxins so that neuromuscular junction is one place that toxins have evolved to to incapacitate the structure itself um is affected in many cases by myotoxins that are based on a phospholipase scaffold but there are also some very specific ones and dr hayashi may not want to discuss this a little bit further but myotoxin a and protamine are these peptide toxins that are very very potent inhibitors of calcium pumps and perhaps a couple of other target sites in muscle cells and specifically in the sr and result in technic paralysis very very rapid well snakes are not satisfied to target just neuromuscular junctions and muscle function but in fact the circulatory system not surprisingly is also a site where we can have structural damage occurring to the vessels themselves we can have cells such as the red cells subjected to damage leading to hemolysis we can have liberation of particular kinds of blood pressure regulators such as bradykinen and many of these calicorn-like serine proteinases are actually producing liberating bradykinin that results in vasodilation there are compounds specifically interacting with platelets interfering with platelet function in a number of different ways these are primarily disintegrants and c-type lectin compounds and then there are thrombin-like enzymes that are very common in viper venoms that in fact will hit fibrinogen directly and affect clotting via the degradation of fibrinogen finally then there's some specialized primarily metalloproteinases but some serine proteinases that can lead to activation of factor 10 and these produce particularly nasty symptoms in humans when envenomated so a number of different targets are different regulatory sites in animals are in fact targeted by these different venom proteins and this is just three common areas that are commonly targeted so what i'd like to do now is take you on a historic journey through some of the things that we've worked with and look at venoms which we can consider bio weapons and how snakes have in fact evolved mechanisms for production and storage safely because if you think about it this gland here containing all these different toxins we just talked about is not very far from the brain and there's a potential then for this toxic arsenal to be problematic for the holder of itself obviously the snakes exist they've diversified so we just need to come up figure out how they're in fact doing this so we've just seen there are a variety of different enzymes in particular as well as toxins that are contained here and in snakes these can be stored for a very long period of time and so snakes at butantan and in my lab some of these we've had for many years some of the snakes don't get extracted and so that venom can stay in that gland for many years in some cases and is ready to go at a moment's notice in addition these snake venom metalloproteinase compounds are in fact autolytic if you try to purify these under the wrong conditions the molecules will chew themselves up so there are problems with storing this venom in this gland that's on the side of the head so how do they do that is venom activated is it stored inactive is it stored in a safe fashion how is it how is that maintained in the gland and are there specific cellular mechanisms that exist among these specialized glands that allow these snakes to do all this have the material ready used in predation perhaps in defense as well but not in fact have toxic effects against the holder of that clan the rattlesnake in this case so if we look at the venom gland itself and this is stuff from way back in the day the venom gland itself is divided into a couple of different regions here and there's a lot of stored venom here and so they can hold that venom in the gland for a very long period of time most of the cells are secretory cells if you extract them there is a very rapid response they increase in height by about 100 percent within a couple of days and they become protein synthesizing factories citrate has been noted as a component of venoms for a long time and we did some work with it a while back and showed that possibly it could be important but in fact in vitro it doesn't at optimal ph for the enzyme but it doesn't actually inhibit a significant amount of metalloproteinase activity citrate doesn't seem to be the most important component here but we're going to come back to that in a moment it turns out there are also endogenous inhibitors and some of these are little tripeptide molecules that are found in the venom oftentimes at millimolar concentrations so they're in in the venom at high concentrations but they're relatively low affinity inhibitors so if we look at some of the metal chelators what we see is that they're very effective at shutting down metalloproteinases and these are pictures from or figures from purified metalloproteinases from a couple of rattlesnakes if we compare the inhibition by these tripeptides what we see is it's much lower affinity for these metalloproteinases so we get out to very high concentrations we can get complete suppression but what it does also do is stabilize them and so if we take the purified metalloproteinase incubate it for a long period of time what we see is we lose activity it eventually drops to almost zero but if it's incubated in the presence of this little inhibitor it in fact stabilizes and retains activity even under incubation at 37 for up to four days or so so very important as an inhibitor so and the stabilizer i should say so the other question is are there cell-based inhibitory mechanisms that occur and what we see is that there's a unusual little cell called a mitochondria rich cell and it occurs in the venom gland at fairly low copy numbers so there's only about two percent of the total population that are these mitochondria rich cells it also cycles with the secretory epithelium as the cells undergo the secretory phase and so they're cuboidal at rest columnar when they're secreting or they're producing and then cuboidal again after they've secreted into the plant and these cells follow that same pattern of increase decrease increase decrease so we're interested in looking at those in a little more detail and it turns out they have some interesting aspects of morphology they sit in this little pocket and in the previous slide we saw you can see a little top of the cell here the apex sticking out like a little button but there's a channel down here that is continuous with the outside that these regions of catalyst that you can see at the arrowheads here and right along here in this space here and when i first saw these years ago i thought this was damaged because i did a bad job preserving the tissue but it turns out those are actual well it turns out those are very very similar in their overall morphology to cells that are found in a rather different sort of place and those are the parietal cells of the gastric pit of the stomach of either mammals like ourselves or rattlesnakes and what we see is this morphology where it's a recessed cell and it has lots and lots of mitochondria very different from the mucous secreting cells that are in the vicinity in the stomach or the serous secretory cells that are typical of rattlesnakes and so that led us to think that maybe we've got something unusual going on here with these things maybe they are specialized and so histologically we did some work staining these tissues and what we see is that these cells here seen here with hd and then here with nitro bt that's specific for these acid secreting cells shows a very localization of these types of activities okay so that was how far we got a number of years ago and it turns out that if we look at the venom ph what we see is that the venom is actually produced at and stored at a moderately acidic ph right about 5.4 and when i've talked with colleagues around the world it turns out that other vipers true vipers as well as pit vipers are right about that same level well if we look at that compared to enzyme activities these are a bunch of purified enzymes from the same rattlesnake venoms what we see is that at this ph there's almost no enzymatic activity well fast forward then into recent times and our collaboration with todd casto's group in texas has led us to be able to look at the same system and now in a lot more detail and what we see here in this diagram is that when we have venom or venom glands that are going from rest to one day post-extraction not surprising we see gene expression of all these different toxin families and they're three listed out here and then combined other minimal ones here but we see a tremendous increase in gene expression coding for those particular toxins not too surprising here okay if we compare that to various other types of tissues though we see a very different sort of pattern okay and so in this heat map what we see is if we look at all of these different transporters here so now we're narrowing our view just onto what's going on with particular gene expression patterns what we see is that in the venom glands you see very high expression in a number of different types of tissues or sorry a number of different types of transporters and these here are the ones that are comparison of unextracted versus one day post-extraction and so what we see in this region here is very hot colors indicating very high levels of expression well we wanted to look at that in a little bit more detail and so we actually looked at some of the specific transporters and asked the question are these being expressed at high levels in these various tissues and so we first looked at gastric membranes from rattlesnakes from rats and from the venom gland just total membranes here and asked the question do we see hk atpases being overexpressed and basically what we see is that they're not expressed so that doesn't look like it's the case this particular transporter here and this is involved in acidification of particular tissues this is not involved in the whole process if we look at a different type of transporter and this is a vacuoler atpase what we see is that just like in the gastric membrane in the venom glands we see that it is very highly expressed and so looking at these western blots provides us with evidence that we have a particular type of transporter that is involved in acidifying the venom could be involved so this looks like a pretty good one to follow up on so we did some fluorescence microscopy on this tissue as well if you remember that little mitochondria-rich cell that was recessed and kind of triangular shape what we see is here and here we see it lighting up specifically with these antibodies designed to recognize those bacular atpases and in all of those cell types only and not in the surrounding epithelium we see those cells lighting up very brightly okay so what we have then is a picture where these mitochondria rich cells that were enigma for many many years to functional morphologists we suggest that they are actually acting as an acidifying cell that is producing acid to drop the ph in the storage conditions of the gland to about 5.4 citrate that has a marginal inhibiting effect we don't think is overly important as an inhibitor directly but it turns out that one of the pkas of citrate is right at about 5.4 and so that means it's going to be a very good buffer for maintaining that ph so we believe that these vacuolar atpases are in fact responsible for acidifying it and the effect is to store the enzymes that potentially could show up with other components and maybe damage the gland in the sink itself are stored at this inactive state further we have spontaneous activation when we inject it into prey because now we're going from a milieu that's 5.4 to a ph two log steps up and so in fact most of the enzymes are very active at 7.4 well like any biological system that's potentially hazardous there are redundant mechanisms for maintaining this and so what we believe is happening is that this is a proximal mechanism that allows for the venoms to be expressed and stored for many years in a stable environment the peptide inhibitors those tripeptides also contribute to this whole thing and then there has been some work done a long time ago showing that some of the metalloproteinases are expressed initially as zymogens but they seem to be clipped shortly after secretion from their cells so this is probably not a major mechanism for safe storage the thing to me that was very interesting is that morphologically and functionally the cell type that's found in the venom gland is very similar to what we find in the gastric pit in the stomach of many different types of vertebrate animals and to me that just indicates another link here between the trophic roles of venom how venoms are used for feeding and digestion and so it's kind of an interesting little aside there that these roles seem to be again conservatively using the same kind of mechanisms for doing particular things a difference here though is that instead of using hk transporter which we see commonly in the gastric pit we have instead this vacuolar atpase as a major transporter resulting in acidification again limited to those mitochondrial rich cells so [Music] well what i'd like to do now is take a look at kind of ecological roles of venoms and also talk about how that has in fact been important towards the eff to the evolution of a particular family of of toxins and that is the three finger toxins so we know that snakes use these venoms to incapacitate prey so here is a a sonoran coral snake eating a little brown snake here and here is a rattlesnake you may not have seen it it's rather cryptic and right there is an animal that's dispatched a little thing called a chipmunk and in fact it surely thereafter consumed that so the venoms we know are important for doing this stopping prey immobilizing prey allowing the snake to handle it okay but the venoms are not the same across the board we've seen that in the intro type of materials that we talked about earlier so what we get are variations of genes occurring via believe first gene duplication these are over expressed in the venom gland and the variety of people including our collaborators have shown this over and over we can get sub functionalization but more importantly neo-functionalization of some of these duplicated genes and this then can lead to expansion of gene families giving us a wide variety of different options that we see in the venoms of various snakes so if we look at particular species we may see as many as 10 to 20 or more transcripts for three-fingered toxins or serine proteinases or things like that so we know that some of these gene families are extremely abundant that is they it's expanded tremendously okay well we talked about this already that there are a number of targeted systems and one of these are the neuromuscular junction in particular i want to look at this one here the acetylcholine receptor which is post-synaptic because this is where a number of different three-finger toxin sub-family groups have in fact targeted for knocking them out okay so the acetylcholine receptor in one form or another is found in all animals major major importance for neuromuscular communication and overall muscular function and toxins typically then will bind to the alpha subunits and incapacitate this structure and the receptor is no longer functional the result is that we get flaccid paralysis of the prey because the neurons are saying contract contract but that signal is in fact blocked okay well let's look at those three finger toxins they're kind of smaller molecules so as far as diversity of toxins are relatively small they're tightly tied together by intramolecular disulfides shown in gold in this diagram here and there are four or five of these typically the fifth one is down here in loop one and that gives them that characteristic canonical structure the three fingers that are tied together at the head group right here okay we find these pretty broadly distributed across various species of snakes the typical activity that most people have focused in is as curare memetic toxins alpha neurotoxic activities binding to that acetylcholine receptor as we've mentioned that is uh skeletal muscle and leading to this flaccid paralysis but it turns out this is one of those scaffolds that has been very very useful for a variety of different types of pharmacologies and so if we look across the board we see a whole bunch of different things and below this line here if you happen to be a salamander this type of molecule is used as a receptivity pheromone and also as a modulator of limb regeneration salamanders can re regenerate limbs so this is a very very useful molecular scaffold and various life forms have in fact played with it to do a wide variety of different things toxic activities or very important activities here involved with modulating structures and reproductive activities okay these have also in fact resulted from gene duplication there have been a variety of different ways attempts to try to explain what's going on it almost seems like there are like cassette like movements of material of genetic material this so-called asset or segment switching in exons and point mutations then after we have gross level changes can fine-tune these kind of interactions okay what we see is that the structurally important residues tend to be highly conserved maintaining that three-finger motif and three-finger morphology but surface residues that are involved with ligand recognition and binding are in fact basically open to variation if you happen to be involved in snake bite treatment these are most problematic because these things bind really really tightly often in the sub nanomolar range and so they basically knock out these receptors and the receptors have to be turned over they're small so they diffuse or they're transported very very rapidly through systems they're often found at very high concentrations and in cobras for example we find that the venom in these two common species indian cobra and monocle cobra may account for 15 to 20 percent of the total venom protein proteome and this is a single toxin not a toxin family but a single toxin cobra toxin untreated of course this often leads to fatal conditions often by respiratory paralysis so there's been a lot of interest in these particular toxins because they're problematic in many parts of the world where lapids are very very common so we see these things in a variety of different snakes and so here is a monocle hook monocle cobra here is a sea snake one of the laticotta species and here is a rearfang snake and what i'd like to do is focus in on some of the toxins from these guys okay so these are three finger toxins we have three over lane here superimposed cobra toxin from cobras full gemotoxin and demo toxins both from different roofing snakes and what we see is that their overall fold is very very similar if we look at their toxicity to mammals sure cobra toxin is very very toxic but if we look at a number of these from different species of rear-fang snakes we see that they're essentially non-toxic and most pharmacologists have stopped here these are not interesting because they don't have much effect we were more interested in them from a biological perspective and how the snake is using these for the prey and so we looked at their effect on other animals non-model animals they're essentially non-toxic to mice but in lizards in this case a gecko this toxin is exceptionally toxic and in small birds they're very very toxic and approximating the toxicity of what we see in cobra toxin so here we have an enigmatic situation non-toxic and mammals highly toxic in lizards and birds and we've seen this repeated in both dimeric and monomeric toxins from a number of different species most recently we've worked with one that also has a variant here and it's got some structural differences from the other ones and it in fact shows the opposite pattern that is it's non-toxic to lizards but it's in fact moderately toxic to mammals these are found in the same venom the amazon puffing snake it's below the sulfurous okay so we're interested in trying to figure out how that actually occurs i'm getting a little bit short on time here so i'm going to move through these a little bit more rapidly structurally they're very very similar but in fact what we see is that if you look at the overall sequence they don't share much of that for forty or fifty percent identity and there are particular areas that we've identified in these taxon specific toxins that affect lizards but not mammals and in fact they are highly conserved in all of these different toxins so in the one that shows that reverse situation what we see is that we have a substitution in one of those invariant motif regions and in the tip we also see a deletion and so there's some structural differences here that lead to overall differences as you've seen colored here these are all highly toxic to birds and lizards but are non-toxic to mammals this one is in fact toxic to mammals but non-toxic to lizards i'm sorry may i ask you a question yes what do you mean about toxic it's thoughts little or is toxin for some specific effects lethal toxicity is what we're looking at here okay is that our problems sorry it's kind of animal well the the ones that we have from calibrates primarily are non-toxic to to mammals but very toxic to lizards and birds okay i was going to ask if there is a big difference between birds and rodents well yeah it's it's an interesting feature that we haven't completely worked out yet as far as what it is that is producing this toxicity what we see though is a very very high level of conservation of the overall scaffold but diversification of function towards toxicity towards particular prey types okay and it turns out that if you look at the animals that the snakes are feeding on these tend to be the highly preferred prey items so they're feeding on birds and lizards whereas things like rattlesnakes and cobras may be also feeding on mammals okay this one in particular is an interesting one because it it has a very stripped down venom and the proteome consists of only well majority of the proteome is three finger toxin rich it's got a couple of different types that are taxon specific towards either mammals or towards lizards it's not a constricting snake and so it's using its venom to incapacitate these two different types of prey so hopefully over the last forty fifteen minutes i've convinced you that venoms and venom glands are an interesting model system to look at a whole bunch of different types of biological phenomena including gene regulation secretion and storage of these bio weapons these molecules are very toxic they're rich source of materials for study and stuff structure and function and with the three finger toxins in particular that is an important component i would argue that understanding how three finger toxins have evolved and diversified in order to do that we're going to have to understand what's going on with these rear fang snakes and only a handful of those have actually been looked at in any detail dimeric structure i have a student that's finishing up his work on this he's starting to get at answering how important the dimer is composed to as opposed to a monomeric structure the nice thing is that if you're interested in this there are over 500 species of rear fang snakes that still haven't been explored at all globally so regardless of where you are around the world there's an opportunity to make important contributions so i'd like to acknowledge support from a number of different funding agencies that have provided us support over the years and of course collaborators have a variety of types cassie modal in particular doing a lot of the molecular work in conjunction with seth fritz and mirnalini very important students and collaborators here todd casto pictured here in the field and his graduate student blair perry absolute tremendous genomics wizards i've had a number of students over the years that have contributed to various parts of these studies as well and my current group is here with a couple of people that weren't there for the picture and so all of their help has been of course tremendously important for that just a little advertisement and dr hayashi has contributed a chapter to this book that is due out very very soon and with that i'd like to thank you for your attention and if we have time i'd be happy to try to answer any questions that you might have so thank you for your attention [Music] thank you very much for your presentation stephen very exciting and very nice pictures well thank you so i do have a question for the audience asking if it if if it is possible that the venom is used for predat pre-digest decree some extent is helping for the digestion of the prey and not only for killing or you know well that's that's a very good question because that was basically the whole focus of my master's thesis and at that time the hypothesis that i was working with was that it is very important to pre-digesting the prey since that time there have been a number of studies looking at the level of digestion and of envenomated versus non-envenomated prey and the suggestion from those studies and there's been several is that the presence of those digestive enzymes is not facilitating digestive efficiency now there are some limitations to the way that those studies were constructed with how you interpret that what we do know is that if an animal is bitten by a rattlesnake or another species that has high metalloproteinase activity there's a lot of structural degradation in addition to the death of the animal that eventually results tissue is broken down so it's hard not to believe that the metalloproteinases and some of the other components are contributing to pre-digesting or tenderizing or conditioning the prey to make it easier to digest okay but that may not be reflected in these metabolic rate studies that were in fact conducted that suggests that venom doesn't have an effect so so i think the i think it does probably have a very important effect but the jury is still out yeah you know you you are focusing on metalloproteins right i do have my own opinion uh actually while i was in bhutan i was working with those very kindly potentiating peptides the bpps yeah actually they are not produced only in the venom so i don't know if you have read some of my publications but i assure you that the any bpp precursor protein can be expressed in other tissues physiological tissues of the snake so for the brazilian pit viper the raka i have seen that the the same rna is expressed or is present in the brain and also in pancreas and for a while i was believing that maybe those bpps in pancreas was somehow symbolizing or being analyzed by by the venom after the digestion so i think that there might have some endogenous physiological function for all those toxins and they are not taught only because they are under expressive so in the glenvein and they are just overexpressed and then when you have a high amount of anything it can be venomous yeah no that that's one of the truisms that we see in venom compositional analysis is that there are analogues or paralogs or homologues found in a wide variety of different types of tissues and and a number of the publications that we've had with todd casto's group have shown the expression across the board of these things but particular subclasses of metalloproteinases of probably some of the bpps as well and certainly things like myotoxins and and other compounds both the pla2s and the peptides are way over expressed in the venom gland relative to their expression levels and other tissues so but yeah it's a it is a common feature that we see across the board that these these toxins are not newly evolved toxins out of nowhere but instead they're co-opted regulatory compounds of a wide variety of different types and that includes even the three finger toxins and so you know those were probably before the evolution of venoms which was probably a very very long time ago hundreds of millions of years ago these were physiological compounds housekeeping compounds and so yeah so it's not surprising we see them in in various tissues yeah so actually my point of view is that even those three peptides that you claim that could be inhibitor or stabilizer from pyroglu um asparagine and some and and they are also actually usually the any terminal of those bpps and i know that they are freely present in the venom so my point is that actually if they do have those same peptides in physiological system they might have a receptor so maybe those are the small peptides present in the venom once they get into the stomach of this steak they are going to synthesize something else acting on a specific receptor so this can trigger some release of digestion liquid or whatever internal enzymes and not exactly about the in the mpps presence individual so are you are you thinking that they would be transported out of the prey into the snake tissues yeah so actually in snake tissue they are going to have those receptors because they are already expressing those peptides yeah so then every activation of something finally in this snake that can you know tell the snake that they are eating something yeah well that certainly could be the case um if the venom though is is internal it's going to take a while for it to break down through the tissues and the stomach acids and digestive enzymes to break through the tissues and allow that to get into the tissues of the snakes so there would probably be a delay of maybe even 24 to 48 hours before that happened but yeah that that could be that then is important as signaling upregulation of of some digestive process or something like that okay i'm sorry i get so excited with your presentation and they do have so many questions but i have to connect to this so there was a student asking why some species of animals are not affected by the venom of other snakes well that's a very good question too and there's i've i've got a student right now that's looking at resistance to venoms in mammals and in in other snakes and a number of people in brazil have actually addressed those same kind of questions and some of them are actually contributors to the book as well um in some respects we can think of uh there's a evolutionary arms race or coevolution between predator and prey and because we're dealing with things that are operating physiologically biochemically we can have a physiological response that can counter the effects of those toxins and we have in we being vertebrate animals whether you're humans or mice there are a number of endogenous inhibitory compounds and alpha macroglobulin is one of them that are basically taking care of what i call rogue enzymes so for example the way that the blood clot is clots is highly regulated by a whole series of cascading steps and you want those to be very active locally but shut down throughout the system so that we're not throwing clots all through and so part of that is regulated by endogenous compounds like that that are in the blood and so what seems to be happening is some animals have the capacity to overexpress some of these compounds they may also be subject to selection favoring modifica modification of their binding properties so that they recognize particular metalloproteinases or serine proteinases or maybe even three-finger toxins and bind them and provide some immunity to to the animal so in the case of the go ahead no you can't finish sorry i was going to mention in the case of three-fingered toxins these are binding specifically two receptors that are found in one whole category of prey lizards and birds versus a very different type of vertebral animal the mammals and precisely how that's occurring we have hypotheses but we're not really sure so okay i'm sorry for interruption okay the other question is from olivia maria but i'm not really sure if you will understand the question so if it's not clear we can just ask her to to ask again but the question is what kind of bioinformatics tools are used to study the potential application of enzyme and other compounds in the pharmaceutical can you recognize those that you can use well i i guess the the big disclaimer is that my bioinformatics background is very minimal and so i rely on the expertise of my collaborators cassie model was a phd student working with me then went to dr kinney's lab in singapore and she she became a very good bioinformaticist and so those kind of questions i largely left on her desk so as far as applications of some of these toxins to potential pharmaceuticals we've we've done a little bit of molecular modeling and such you know in silico um but not a not a real extensive a lot amount of that again i have a phd student that is hoping to finish up soon and he's interested in doing some of that work so yeah okay i hope olivia is happy with this sensor if not please write question again so yeah the question is uh from one david and he says thanks for your awesome presentation and he was like you if we you think that uh new previously only unintentified protein is careful so still unknown protein is careful might may still be hidden in veno from here fanged snakes i don't know why this is a specific snake but anyway yeah so are there are there some novel scaffolds out there that we haven't yet discovered yeah and they could be useful for magical bias that exist towards vipers and elephants so i think that his concern his concern is about some specific snakes because there is a lot of elephants and vipers but not so many about other snake species yeah well i think that the the colubric snakes globally the rear fang snakes are still a very productive area for looking for new compounds and interesting compounds but one of the things that is apparent is that snakes are fairly conservative with the selection of things that they've really grabbed onto and diversified okay so the types of compounds that we see as the most predominant in any particular venom you see the same basic players again and again and again now there are lots and lots of compounds in the venoms that are expressed in very very small amounts and from a perspective of what is the biological relevance of some of those compounds they're probably not important to producing the symptoms of infantimation it's possible some of them are biologically you know super active highly active but it's unlikely that they're contributing as much if they're only 0.01 percent of the total protium as something that's twenty percent for example just to give you an example of of the differences in our part of colorado the prairie rattlesnakes venoms from adult snakes from a den site that we've worked at the myotoxin a component of those venoms can comprise excuse me up to 35 percent of the total venom protein just that one particular molecule and so they they've really heavily invested in that direction if you will evolutionarily so um so i think we're going to see a lot of the same kind of molecules same kind of scaffolds as the abundant proteins now if you're interested in mining the less abundant proteins and really trying to identify those that's going to require some kind of cloning and over expression system in order to work with the material pharmacologically but that could be potentially very very interesting and illustrative and as as far as novel compound leads that could be more productive than going in and asking what are the dominant components of this venom okay because it seems like we're seeing the same kind of things showing up in snakes whether they're rarefang snakes from africa from south america from north america or alapins and vipers from other places around the globe we're getting differences in the overall abundances of a variety of different proteins but we're seeing the same kind of players as the major players in the venom again and again okay yeah so if you allow me um in my point of view those with a very low concentration in the venom can be obsolete fine if they have a very strong pharmacological effect so it's a very specific biological effect that you can trace then it's make easy to you didn't find it and then we can express by recombinant or using synthetic biology because there are different ways and now speaking about the composition of the venom by itself some of the toxins like crotamine it can represent up to 25 percent of the dry weights of the vena but even though it's not really important for the envenomation process so quite so eminent do you have any i'm sorry you said it's 25 of the venom but it's not important for invention yeah it's not that important you will never see any rodent or a right or mouth with the paralyzes paralyzed because of the croton you can just see it after purification not using the approved venom it does not be important for the animation but it is important for for the accident human accidents so if you use a vaccine against a venom without scrutiny then you cannot protect it uh foreign but for the venemation by itself for the prey i have never seen any specific effects biological this is native species of prey uh yeah i'm talking about rats and mice inbred inbred strains or or wild wild animals um actually working with the experimental animals in the lab nothing but uh even for wild animals if you just observe the nature they are not designed of the paralysis they are usually dying because of phospholipases it's a neurotoxic effect yeah well that venom obviously has that pre-synaptic neurotoxin too i know that in our prairie rattlesnakes when they bite a lab mouse that that lab mouse is paralyzed almost instantaneously and so for them that's very very important um i'm surprised that crotamine in lab mice doesn't have that same effect because the sequence is almost identical yeah i mean i'm talking about crude protamine p peripheral protein has affected that in maybe animals with different sensitivity dependent of the strain i'm talking about you know if you use the crude venom yeah it does not make any difference you don't see clothamine effect using protein so you don't get hyperextension and immediate paralysis no it dies before due to the neurotoxin effect of phospholipids yeah that's curious yeah like i say with the crude venom we don't see that yeah anyway i will go back again for the uh apologies because there is so many questions one of them institute and she's just uh an excellent presentation congratulations there is from juan gal gutierrez and do you think venom could display some degree of phenotypic plasticity on response to environment pressure for example prey ability availability so if for example if you have a single snake and it feeds on this type of prey or that type of prey i suppose yes if we wanted to improve this question we'll be happy yeah well there's been a lot of discussion about whether or not you can have this kind of epigenetic effect modifying the venom composition now we know that venoms do change in composition for many species when they're newborn to adult and their compositional changes that can be pretty profound but from the limited amount of work that's been published and from a little bit that we've done that hasn't been published if you feed lizards to one group and mice to another group you don't see a very profound effect there's one paper that was published that showed in one snake that there was a tendency towards over expression of phospholipase i think in in the mammal fed group but other than that the effect on the individual snake doesn't seem to be particularly profound regardless of the type of food so that epigenetic effect or the regulatory effect on it directly there doesn't seem to be a direct feedback there but there's another way to looking at these right like him we have the uh pit viper but both representative that's that version of those bpps these incontinator area they have a different composition of the venom compared with the both tropes that live in ireland like both ups in slime yeah yeah it's birds therefore somehow what they are eating are influencing the composition of the venom don't you believe in that it's like yeah you're right i agree all right that's true the chronology in brazil the right here has a specific composition that's closer to both raps in north america than rotolos in north america the composition are completely different though so they are 18 different stuff no yeah i i think there is a direct association between venom composition and prey type okay but i don't think that snake an individual snake has the capacity to change the expression pattern of its venom if it's fed this birds versus mammals or something like that okay that doesn't be occurring yeah it's a a kind of long-term adaptation yeah but seeing just one single individual but that's true even just single individual if you maintain that captive in captive they are going to change the amount of uh they they produce spending their sex and yeah in captive since the heading it seems that the toxin amounts in the venom and also the amount of the venom that is produced is different compared when they are in the nature well adaptation yeah i'm not sure that i agree with that 100 i think things do change a little bit but from our experience at least snakes that we've had in captivity for a long period of time for a short period of time they show almost no difference for a long period of time we see very little difference in extractions of a snake that were taken 10 years apart so so to me that that suggests that the effect of captivity and feeding regime that you've imposed on the snake and now can't feed on whatever it would eat in the wild doesn't have an a major effect on venom composition as long as it's nutritionally sufficient not not starving or something like that yeah it might have some also effects of the snake type the species so depending on your skin faster and the other does not care if they are in captive or not but in addition to this question there is another question from natalya so she's asking if he'll make venoms present prostitutes plasticity or could adapt to the prey on an ecological level and if these features have been seen only in the evolutionary way does these adaptable uh futures of plasticity is only for evolutionary way or it can present some adaptive adaptation or plasticity could could adapt to the place or on ecological level if it's possible to have the change adaptation of the snake yeah you know yeah i think it is but i think one of the basic questions is how fast can venom composition change right and what are the factors that affect it and there have been a number of people including us that have tried to look at that question and um it's it's interesting that a lot of different factors seem to be minimally important there's some studies that suggest that things like overall environment and climate may in fact be linked to compositional trends and shifts and things like that um but there's there isn't i haven't seen any studies that have really been here this defines this is the factor that shapes it directly it's it's more kind of a much softer kind of a effect that is it's it's not a black and white effect that you know if if you eat this prey and shift over to this then the venom composition will change in a particular direction so so i think yeah go ahead no it's just going to say that i think these kind of things over evolutionary time we do see these kind of transitions the problem of course is that we're not long long-lived enough to track these things over a long period of time and that's that's one of the limitations yeah it seems that this matter really um attracts a lot of attention so there's a couple of more questions adaptations so we skip them and i would like to ask you if we can just go go back to your slides and show your new book so the people oh yeah making a kind of advice for you yeah so please guys buy this book so you're going to to find out our answer for your questions and i'm pretty sure that the dr stephen is ready to answer any further questions you have so you can just send to me and i can afford for him and for later we also shared the recording of this scene so you can watch it again anytime you want so on this point i would like to thank dr stephen for accepting my invitation for this uh awesome presentation it was very exciting a lot of questions so if you want to give some last words just take your time yeah okay well i'd be happy to answer any questions again if i can and if you have questions that come to mind later today tomorrow feel free to drop me an email i'd be happy to answer anything like that too so yeah now it's been my pleasure i my only regret is that i'm not there in person because i would love to be in sao paulo and see your university and visit with you face-to-face so actually you are already invited you can come anytime you will be welcome we just have to get off of this academic situation yeah and i'm i'm concerned that this is going to last a lot longer than any of us want and any of us are predicting but hopefully hopefully things will get better you were entering winter time or autumn i guess and and that's a there's a possibility that uh severity will increase a little bit you know we've we've hit a situation where caseload has dropped a little bit but starting to spike back up again so it's hard to predict but i would love to visit again as i mentioned i've only been to sao paulo once i did experience though your traffic jams there um dr enacio guevara actually took me out to the beach at the atlantic coast and on the way back i think we were stuck in traffic for almost 12 hours to return so really i never reminded me of southern california i'm always in the in the opposite way so where i know there is going to be i'm pointing the opposite way so you go to the amazing summer and you go to the beach in winter that's the best way yeah never do the same as everybody otherwise it's awful it's uh i try not to but sometimes you can't help it so next time you come i'm quite good for skipping uh traffic okay i'll take you up on that then okay so thank you again let's just start again my pleasure yeah and we can keep discussing and maybe you can introduce me mark your friend mark thomas was that sure yeah i will try to do that